López et al. Virology Journal (2020) 17:63 https://doi.org/10.1186/s12985-020-01327-9

SHORT REPORT Open Access First report of Lihan Tick virus (Phlebovirus, Phenuiviridae) in ticks, Colombia Yesica López1, Jorge Miranda1, Salim Mattar1*, Marco Gonzalez1 and Joel Rovnak2

Abstract Background: Tick-borne phenuivirus (TBPVs) comprise human and animal viruses that can cause a variety of clinical syndromes ranging from self-limiting febrile illness to fatal haemorrhagic fevers. Objective: Detect Phlebovirus (Family Phenuiviridae) in ticks collected from domestic animals in Córdoba, Colombia. Methods: We collected 2365 ticks from domestic animals in three municipalities of the Department of Cordoba, Colombia in 2016. Ticks were identified and pooled by species for RNA extraction. A nested real-time PCR with specific primers for Phlebovirus and a specific probe for Heartland virus (HRTV) formerly a Phlebovirus, now a Banyangvirus were performed. Also, a conventional nested PCR, with the same specific primers was used to detect other Phleboviruses, with positive reactions indicated by an amplified cDNA fragment of approximately 244 bp determined by gel electrophoresis. These bands were gel-purified and sequenced by the Sanger method. Results: Using real-time RT-PCR, no positive results for HRTV were found. However, using conventional nested PCR 2.2% (5/229 pools) yielded a product of 244 bp. One positive sample was detected in a pool of Dermacentor nitens ticks collected from a horse, and the four remaining positive pools were from Rhipicephalus microplus collected from cattle. The five positive nucleotide sequences had identities of 93 to 96% compared to a section of the L- segment of Lihan Tick virus,aPhlebovirus originally detected in R. microplus ticks in China. The strongest identity (96–99%) was with Lihan Tick virus detected in R. microplus ticks from Brazil. Conclusions: This is the first report of viral detection in ticks in Colombia. We detected a Colombian strain of Lihan Tick virus. We recommend expanding the sampling area and carrying out more eco-epidemiological studies related to epidemiological surveillance of viruses on ticks in Colombia. Keywords: Phlebovirus, Tick-borne diseases, Epidemiology, Zoonotic

Synopsis Background Phleboviruses are RNA viruses transmitted by ticks, Members of the family Phenuiviridae include human and phlebotomines and mosquitoes. This study presents the animal pathogenic viruses transmitted by arthropod first detection of Phlebovirus (Lihan Tick Virus) in ticks vectors, including phlebotomine sandflies, mosquitos and from Colombia. The sequences detected are related to ticks [1, 2]. They can cause a variety of clinical syndromes Lihan Tick Virus previously reported in Brazil and ranging from a brief, self-limiting febrile illness, to retinitis, China. encephalitis, meningoencephalitis and fatal haemorrhagic fever [3]. The genus Phlebovirus belongs to the family Phenuiviridae,orderBunyavirales, [4]. The genus includes * Correspondence: [email protected]; 10 species with around 58 viruses and several that have not [email protected] 1Instituto de Investigaciones Biológicas del Trópico, Facultad de Medicina yet been categorized [5]. Phlebovirus genomes are single- Veterinaria y Zootecnia, Universidad de Córdoba, Montería, Colombia stranded RNA with negative polarity, organized into three Full list of author information is available at the end of the article

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. López et al. Virology Journal (2020) 17:63 Page 2 of 6

segments. Segment L is approximately 6.4 kb and encodes a warm tropical climate, an average temperature of an RNA-dependent RNA polymerase, segment M is 28 °C, with altitude ranging from 0 to 40 m above sea approximately 3.4 kb and encodes viral glycoproteins, and level. Its principal economic activity is livestock, agricul- the S segment is approximately 1.7 kb and encodes the ture and in grazing, which is over exploited, affecting nucleocapsid protein. Additional non-stuctural proteins ecosystems and increasing the burden of ticks potentially (NSS) are encoded on the S and M segments [3, 6, 7]. responsible for the transmission of many zoonotic dis- In 2018, TBPVs were divided into four phylogenetic- eases [18]. ally related groups: i. the Uukuniemi group, which includes at least 17 species of tick-borne phleboviruses; Ticks collections and taxonomic identification ii. the severe fever with thrombocytopenia syndrome Hard ticks from the family Ixodidae were collected at virus / Heartland virus group; iii. The Bhanja group; and all developmental stages by removing them from their iv. the Kaisodi group. Most of these viruses cause disease vertebrate hosts. For storage and transport, ticks were in humans [4]. placed in small containers with porous caps for oxygen However, in 2019, the group of severe fever with exchange. Ticks identification was performed using the thrombocytopenia syndrome virus / Heartland virus, was taxonomic dichotomous keys [19]. Once taxonomically taxonomically reclassified in the genus Banyangvirus [5]. identified, they were frozen at − 90 °C. Ticks were char- In recent years, new TBPVs capable of inducing serious acterized by species, sex and stage and pooled by spe- diseases in humans have emerged. cies, municipality, date and host. The prevalence of In 2007, the first cases of severe fever with Phlebovirus in ticks was expressed as a minimum infec- thrombocytopenia syndrome virus (SFTSV), was found tion rate per 1000 ticks (MIR) based on the assumption in China, later were reported in Japan, Korea and that a PCR-positive pool contains at least one positive Vietman in patients with severe fever, thrombocytopenia tick [20]. and leukocytopenia accompanied by gastrointestinal Phlebovirus detection RNA extraction was carried symptoms, chills, joint pain and myalgia [8–12]. In the out with PureLink® RNA Mini Kit combined with TRI- western hemisphere, Heartland virus (HRTV) was first zol® (Invitrogen). RNAs were reverse transcribed with isolated in 2009 from two patients in the state of Mis- Moloney Murine Leukemia Virus Reverse Transcriptase souri, USA, who showed symptoms similar to those of (M-MLV RT), random primers and amplified with SFTSV. HRTV is phylogenetically related to SFTSV with nested PCR primers designed to amplify conserved a similarity of 60 to 70% at the nucleotide level [13]. Phlebovirus L segment sequence. Amblyomma americanum ticks appear to be the main For the first round, a conventional PCR was carried vector for HRTV transmission. To date, more than 30 out with the genus-specific primers: NPhlebo1f 2054- cases of HRTV infection, including two deaths, have ATGGARGGITTTGTIWSICIICC-2074 and NPhlebo1r been reported in the United States [14–16]. The status 2295-AARTTRCTIGWIGCYTTIARIGTIGC-2273. For of HRTV in subtropical and tropical regions of the the second round, real-time PCR was used with genus- Americas remains unknown. specific nested primers: NPhlebo2f 2027-WTICCIAAIC- In Colombia, ticks of the genus Ixodes, Dermacentor, CIYMSAARATG-2049, NPhlebo2r 2578-TCYTCYTTRT Amblyomma, Haemaphysalis and Rhipicephalus could TYTTRARRTARCC-2552 [21] and a FAM-labeled Taq- act as vectors for a variety of viruses including TBPVs manTm probe 5′-CGAGTGGAATGGACAGGGAC- [17]. However, there have been no studies looking for BHQ1–3′ for specific detection of HRTV. HRTV RNA viruses in ticks to date. The aim of this study was to was used as positive control. In addition, for the second detect and identify HRTV and other TBPVs in ticks col- round a conventional PCR, with the same specific lected from domestic animals in different municipalities primers was used to detect other Phleboviruses. of the department of Córdoba, Colombia.

Methods Sequencing and phylogenetic analysis Type of study, geographic area and sampling sites Positive samples were subject to direct sequence of both A descriptive and prospective cross-sectional study was strands in independent reactions with each of the conducted between June and December 2016. Ticks internal primers and analyzed by the Sanger method attached to dogs, horses, and cattle were collected in the (Quintara Biosciences, Inc., Berkeley, CA, USA). Nucleo- municipalities of Montería, Cereté, San Pelayo and tide sequences were subjected to a BLAST analysis to Puerto Escondido in the department of Córdoba, identify homologous sequences in GenBank. Twenty-six Colombia (Fig. 1). Córdoba is located in the northwest- sequences of the L segment of different Phleboviruses ern part of Colombia from 7° 22′ to 9° 26′ north latitude were downloaded from GenBank and aligned using and from 74° 47′ to 76° 30′ west longitude. Córdoba has MUSCLE in MEGAX. The phylogenetic analysis was López et al. Virology Journal (2020) 17:63 Page 3 of 6

Fig. 1 Location of tick collection in four municipalities (Puerto Escondido, San Pelayo, Cerete, Monteria) of the Department of Córdoba performed using the maximum likelihood method with species (244 bp). One amplicon was detected from 63 Kimura 2 parameters model with MEGAX [22].. pools of D. nitens collected from equines (MIR 1.02; 1/978 ticks tested) and the other four amplicons were found Results among 144 pools of R. microplus collected from bovines A total of 2365 ticks were collected, including 1358 (MIR of 2.95; 4/1358 ticks tested). None of 29 R. sangui- (57.5%) R. microplus collected from bovines, 978 (41.3%) neus ticks collected from dogs tested positive. No positive D. nitens collected from equines, and 29 (1.22%) Rhipice- results for HRTV were found. phalus sanguineus sensu lato collected from dogs. Ticks Alignments of the 244-bp amplicons showed that three were organized in 229 pools. of the sequences were identical and the other two sequences had small changes in the nucleotide Detection of viral RNA in ticks and sequencing analysis sequences of 1 and 7 bases. We found that 2.2% of tick pool extracts (5/229) yielded Sequencing and BLAST analysis showed that the five PCR amplicons with the length expected from Phlebovirus sequences detected have an identity between 93 to 96% López et al. Virology Journal (2020) 17:63 Page 4 of 6

with sequence from the Phlebovirus Lihan tick virus,L- China. These viruses are most similar to other TBPVs segment, originally identified in R. microplus ticks in and are distant from Phleboviruses transmitted by sand- China [23] and identity between 96 to 99% with Lihan flies and mosquitoes. tick virus detected in R. microplus ticks in Brazil in 2018 [24]. Sequences were deposited in GenBank under the Discussion following accession numbers: MK040531, MN026336, Ticks are transmitters of many viruses of importance in MN026337, MN026338 and MN026339. public health. However, viral diversity in ticks from South America is largely unknown [24]. In recent years Phylogenetic analysis the emergence of new TBPV such as SFTSV and HRTV A phylogenetic tree was constructed with the five in Asia and North America, respectively, present a sig- sequences reported in this study and 19 related nificant public health threat [4]. In the present study, sequences available in the GenBank, as well as outlier TBPV sequences detected in Colombian ticks collected sequences from tick and mosquito-borne viruses, form domestic animals were similar to Lihan tick virus Mukawa virus and Gouléako virus respectively (Fig. 2). detected in China in R. microplus ticks [23]. Our results The tick virus sequences detected in this study formed a also agree with a work carried out in Southern of Brazil clade with sequences of Lihan tick virus from Brazil and by Souza et al., in 2018, who analyzed six groups of R.

Fig. 2 Phylogenetic analysis of Colombian Lihan Tick virus. Evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 26 nucleotide sequences. Bootstrap values were deduced from 1000 replicates. There was a total of 149 positions in the final dataset López et al. Virology Journal (2020) 17:63 Page 5 of 6

microplus ticks (~ 50 per group). They used a metage- Ethics approval and consent to participate nomic approach with high-throughput sequencing Not applicable. (HTS) and detected Lihan tick virus in 5 of 6 groups of Consent for publication ticks [24]. Sameroff et al., 2019, conducted a study in Not applicable. Trinidad and Tobago in 2017 and 2018, in which they analyzed 638 ticks, including R. microplus (n = 320), R. Competing interests sanguineus (n = 300) and A. ovale (n = 18) with HTS. The authors declare no competing interests. Lihan tick virus was detected in 14 of 16 pools of R. Author details microplus (MIR of 43.75; 14/320 ticks tested) results that 1Instituto de Investigaciones Biológicas del Trópico, Facultad de Medicina Veterinaria y Zootecnia, Universidad de Córdoba, Montería, Colombia. differ from the MIR of 2.95 (4/1358) reported in our 2Department of Microbiology, Immunology, and Pathology, Colorado State study. However, the methodology used for the detection University, Fort Collins, CO, USA. of the Lihan tick virus in these two studies was different, and Sameroff et al. used 20 ticks per pool, whereas we Received: 4 February 2020 Accepted: 8 April 2020 tested pool sizes of 10–15. Furthermore, the stage and sex distribution of ticks and season of collection in the References two studies may have differed. These differences as well 1. Matsuno K, Weisend C, Kajihara M, Matysiak C, Williamson BN, Simuunza M, et al. Comprehensive molecular detection of tick-borne Phleboviruses leads as geographic variation could explain the variability in to the retrospective identification of taxonomically unassigned Bunyaviruses the results [25]. and the discovery of a novel member of the genus Phlebovirus. J Virol A limitation of the present study is the short fragment [Internet]. 2015;89(1):594–604. Available from: http://jvi.asm.org/lookup/doi/1 0.1128/JVI.02704-14. 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